Epidemiology and genetic diversity of Streptococcus suis in smallhold swine farms in the Philippines

This study aimed to determine the presence and characteristics of locally circulating strains of Streptococcus suis, the most important streptococcal pathogen in swine. Oral swab samples were collected from pigs from 664 representative smallhold farms across nine provinces in the Philippines. Isolates were identified and characterized using PCR assays. The study revealed an isolation rate of 15.8% (105/664, 95% CI: 13.0–18.6) among the sampled farms. Two hundred sixty-nine (269) S. suis isolates were recovered from 119 unique samples. Serotype 31 was the most prevalent (50/269, 95% CI: 13.9–23.2) among the other serotypes identified: 5, 6, 8, 9, 10, 11, 15, 16, 17, 21, 27, 28, and 29. The detection of the three ‘classical’ S. suis virulence-associated genes showed that 90.7% (244/269, 95% CI: 87.2–94.2) were mrp-/epf-/sly-. Multilocus sequence typing (MLST) analysis further revealed 70 novel sequence types (STs). Notably, several local isolates belonging to these novel STs formed clonal complexes (CC) with S. suis strains recovered from Spain and USA, which are major pork-exporting countries to the Philippines. This study functionally marks the national baseline knowledge of S. suis in Philippines.

In total, swab samples were individually collected from the oral cavities of 1,567 pigs, particularly from the surface of the palatine tonsils.Among these samples, 269 S. suis isolates were recovered from 119 unique samples (7.6%, 95% CI: 6.3-8.9).All isolates were recovered from asymptomatic pigs, except for two isolates obtained from a deceased piglet that exhibited fever symptoms before coincidentally dying on the day of sampling.No additional information was available for these two isolates that would confirm clinical S. suis infection.Therefore, these two isolates were characterized together with all other isolated S. suis strains.No isolates recovered from systemic or invasive clinical infections of S. suis were included.
To further validate the identity of the S. suis isolates, 11 representative isolates were subjected to 16 s rRNA gene sequencing.BLAST analysis revealed a 100% query cover and a 99 to 100% percentage identity (PIaD) to S. suis.All sequences were submitted to NCBI with GenBank accession numbers OR287118-OR287128. Figure 2 shows the phylogenetic tree constructed from the partial 16 s rRNA gene sequences of the recovered isolates, alongside several type and representative sequences from other streptococcal species.

Molecular serotyping
From the 119 unique swab samples gathered from the sampled animals, 269 S. suis isolates were recovered.The identification of the capsular polysaccharide (cps) types of S. suis was carried out using a four-reaction multiplex PCR assay 19 .
Among the recovered isolates, 131 of isolates tested were assigned to the 29 recognized serotypes, while the remaining 138 isolates could not be classified into any specific serotype and were categorized as nontypeable isolates.Nontypeable isolates were the most prevalent, followed by serotype 31.The proportion of the nontypeable strains (138/269, 51.3%, 95% CI: 45.3-57.3)was significantly higher than any of the typeable strains, as evidenced by their 95% confidence intervals.Additionally, among typeable strains the proportion of serotype 31 strains (50/269, 18.6%, 95% CI: 13.9-23.2) was significantly higher than all other detected typeable serotypes.Furthermore, the proportion of serotype 9 strains was significantly different from serotypes 31, 27, 5, 6, 29, 11,  17, 10, 28, and 15 but not significantly different from serotypes 21, 16, and 8. Subsequently, the proportion of serotype 27 strains was significantly different from serotypes 9 and 31 but not significantly different from the rest of the typeable strains (Supplemental Fig. 3).
It could also be important to note that 52.0% (95% CI: 32.4-71.6) of isolates positive for VAGs were recovered from sows and piglets.

Multilocus sequence typing
From the 269 S. suis isolates, 145 were selected for multilocus sequence typing (MLST) using seven different housekeeping genes (aroA, cpn60, dpr, gki, mutS, recA, and thrA).The sequences were submitted to pubMLST database, and analyses revealed 101 novel alleles.Specifically, 14 new alleles were found in the aroA gene, 19 in the cpn60 gene, 10 in the dpr gene, 17 in the gki gene, 17 in the mutS gene, 13 in the recA gene, and 11 in the thrA gene.
Interestingly, it was determined that all the 145 isolates could not be typed into any of the previously reported STs available in the database and were subsequently assigned to 70 novel STs.The most common ST observed was ST2157, which was observed in 11 isolates, followed by ST2162, ST2166, and ST2211, each with seven isolates.Meanwhile, ST2168 and ST2181 have six isolates each, and ST2170, ST2176 and ST2215 were represented in the collection by five isolates each.Further analysis of the association between serotypes and STs (Fig. 3.A.) showed that eight out of the 14 identified serotypes exhibited multiple STs.Serotype 31, the predominant serotype, exhibited 22 different STs.These include ST2152, ST2166, ST2167, ST2168, ST2175, ST2176, ST2189, ST2191, ST2192, ST2193, ST2194, ST2195, ST2197, ST2198, ST2199, ST2200, ST2201, ST2202, ST2270, ST2271, ST2272, and ST2274.The number of distinct STs within the other serotypes range from one to 10 (Fig. 3.A).
Notably, two STs from the Philippines were connected to two other STs, one from Spain and another from USA.Specifically, ST2157 from Philippines belongs to CC17 via ST14 from Spain, while ST2161 from the Philippines formed a clonal complex with ST848 from USA.A total of 7.6% isolates (11/145) belonged to CC17 and 1.4% isolates (2/145) belonged to CC848/CC2161.

Discussion
Pigs and wild boars are considered natural reservoirs of S. suis, and colonization is generally regarded as present in almost all herds 1,22 .An individual pig, whether diseased or asymptomatic, may carry a mixture of multiple and diverse S. suis strains colonizing the upper respiratory tract, particularly the tonsils and nasal cavities, as well as the vaginal and possibly the alimentary tract 1 .The transition of S. suis from a colonizing pathobiont into www.nature.com/scientificreports/an invasive pathogen is driven by a complex multifactorial process influenced by host health, environmental conditions, and strain-specific factors 22,23 .
In diseased pigs, examination of the clinical manifestation, the age of the affected individuals, and the characteristics of macroscopic lesions are common reference points for a presumptive diagnosis of S. suis clinical infections.This diagnosis is only confirmed when microscopic lesions typical of S. suis infection are observed, and isolation of S. suis is achieved from tissue samples, preferably from multiple organs or sites 1 .Meanwhile, in asymptomatic pigs, the recovery of S. suis strains from the nasal cavities or tonsils suggests a high load of this bacterium in these sites.This is because microbiological isolation is of low sensitivity for detection due to the presence of competing members of the microbiota.Isolation of S. suis strains from these sites may, therefore, indicate active transmission rather than a carrier state 1,24,25 .However, this distinction has not consistently been applied in the literature such that the proportion of pigs in a sampled population that is culturally positive for S. suis has also been referred to as the carriage rate, while the individual animals have also been called carrier pigs [26][27][28][29] .
It is crucial to note, however, that such colonizing isolates of S. suis from asymptomatic pigs should not be automatically considered avirulent or non-pathogenic strains.The classification of S. suis isolates as virulent or avirulent, pathogenic, opportunistic, or strictly commensal is a complex issue that has previously been problematized and discussed.In brief, there is no standard definition of what a virulent strain is.Asymptomatic pigs may carry virulent strains, particularly taking into account that most human cases of infections are acquired due to the consumption of contaminated pork products that are presumably from healthy pigs.Further complicating www.nature.com/scientificreports/this problem is the absence of standardized assessment methods and models, either in vivo or in vitro, to assess strain virulence or evaluate the importance of suggested critical virulence factors, which would enable direct comparison of results 30 .
As such, for both clinical infections and asymptomatic colonization of S. suis, direct detection of S. suis by molecular detection from tonsils or nasal cavities offer limited practical utility 1,31 .On the other hand, while microbiologic isolation of S. suis strains may be less sensitive than molecular or serologic detection, isolation is a requirement for the confirmation of clinical infection and identification of potentially actively transmitted strains in asymptomatic pigs 1 .Furthermore, recovered isolates, regardless of the clinical condition of the source, offer opportunities for further genomic and phenotypic characterizations, including but not limited to serotyping, detection of virulence-associated genes, MLST, host challenge assays, and antimicrobial resistance, among others.These pieces of information are of utmost importance in the characterization of the epidemiology of S. suis and in the holistic evaluation of the importance of circulating S. suis strains.Microbiologic isolation of S. suis strains, therefore, has been utilized in numerous studies across various countries to describe the prevalence and characteristics of S. suis in both diseased and asymptomatic pigs 28,[32][33][34][35][36][37][38][39][40][41][42] .
Almost a decade after Huong et al. ( 2014) singled out the Philippines as the S. suis knowledge gap in Asia, this study presents a representative cross-sectional survey in smallhold swine farms in the country 12 .No prior information on outbreaks, if they had occurred, or list of farms with a history of S. suis-related diseases was available to guide the selection of farms for sampling.Instead, smallhold farms were sampled from high pig population density provinces in the country.
The observed positive isolation rate among pigs in the current study (119/1567, 7.6%, 95% CI: 6.3-8.9) is comparable to an initial report of S. suis isolation in the Philippines in 2020, which achieved a 9.09% (4/44, 95% CI: 0.6-17.6)isolation rate from a single farm 8 .However, this rate is higher than an older report detailing zero recovered isolates from 220 nasal and tonsil swabs of slaughtered pigs from a province not included in the current study 43 .Similarly, reports on S. suis isolation from healthy pigs from farms or slaughterhouses in other countries, such as Northern Vietnam 44 , China (Jiangsu) 45 , Northern Thailand 46 , and Turkey 43 , showed isolation rates ranging from 0 to 6.0% .On the other hand, reports from China (Anhui) 47 , Thailand (Chiang Mai) 48 , and China (Xinjiang) 47 ranged from 7.8 to 8.3%.Lastly, isolation rates reported from other provinces in China 47 , Korea 49,50 , India 51 , Spain 52 , Canada (Quebec) 53 , Thailand (Phayao) 27,54 , UK 26 , Northern and Central Thailand 28,46 , Southern Vietnam 36 , and Canada (Ontario) 55 ranged from 11.2 to 73.1%, demonstrating a wide range of reported S. suis isolation rates in the literature (Supplemental Fig. 4).
Currently, 29 serotypes of S. suis are recognized based on the serological characteristics of the bacterial capsular polysaccharide [56][57][58][59] .Among these serotypes, serotype 2 is most associated with infections in both pigs and humans.It has been determined to be responsible for over 80% of human cases, putting emphasis on its importance as a global zoonotic threat 60 .As a result, a significant portion of the research and reports has primarily focused on S. suis serotype 2 samples obtained from diseased pigs and humans.Globally, the predominant S. suis serotypes in clinical pig cases are serotypes 2, 9, 3, 1/2, and 7.However, within North America, serotypes 2 and 3 were the predominant serotypes, followed by serotypes 1/2, 8, and 7 3,30,61 .This can be attributed to the fact that the distribution of serotypes exhibits significant variations over time and in different geographical areas 41 .
In this study, no serotype 2 strains were recovered.Serotype 31 (38.17%;50/131) was detected in 7 out of 9 provinces sampled, which may suggest that there is a need to put serotype 31 under surveillance to further understand its clinal and epidemiological importance, and identify potential control measures, if necessary.This is particularly important since other studies in Vietnam, Thailand, Spain, Canada, UK, and China only reported isolation rates of serotype 31 strains ranging from 0 to 7.2% in healthy or asymptomatic pigs 28,26,62 .On the other hand, there have also been several reports of serotype 31 associated with diseased pigs in Canada 42 and China 63 (Supplemental Fig. 5a).Notably, this serotype has also been reported to have caused a human infection in Thailand 56 .
Furthermore, over the last two decades, there has been an observed increase in the prevalence of serotype 9 isolates from diseased pigs in Netherlands 33 , Spain 39 , and Canada 62,64 , and it is also among the most commonly isolated serotypes in both clinically healthy and diseased pigs in commercial farms in China 65 .Reported proportion of serotype 9 isolates from diseased pigs ranged from 0-47.3% as reported from studies in Canada 42,55,62 , Spain 39 , Brazil 66 , Taiwan 67 , and the Netherlands 68 .Meanwhile, studies on healthy or asymptomatic pigs in Canada 55,62 , China 26 , Vietnam 36 , Korea 49,50 , Thailand 27,28,54 , UK 26 , and Spain 52 ranged from 0 to 15.9% (Supplemental Fig. 5b).
The predominance of nontypeable strains in the current study aligns with findings from other publications reporting that nontypeable S. suis strains are commonly isolated in field conditions.These strains may be considered as potential novel serotypes or mutants of known serotypes 19,73,74 .Studies demonstrated that unencapsulated strains, which include nontypeable isolates, exhibit unique properties such as increased adherence to surfaces and cells, as well as the ability to form biofilms, which may contribute to their persistence and transmission 75 .Nontypeable isolates are also frequently recovered from both clinically ill and healthy pigs 74 .Although available information is limited, there have been reports of nontypeable S. suis isolates from pigs with meningitis in China (designated novel serotype variant Chz) 76 , and a nontypeable unencapsulated S. suis strain has also been reported www.nature.com/scientificreports/ in a human case in Thailand 77 .Hence, it is also important to consider potential risks associated with nontypeable isolates, considering that their role in disease transmission could not be disregarded.The predominant S. suis VAG genotype (90.71%; 244/269) identified in this study is mrp−/epf−/sly−.This is consistent with other reports on S. suis isolated from healthy/carrier pigs from China 78 , Thailand 28 , and Germany 35 , forwarding the observation that isolates positive for epf, mrp, and sly genes were significantly less frequently detected in clinically healthy pigs 35 .It is important to note, however, that this determination does not automatically indicate avirulence, since most virulence-related studies that considered epf, mrp, and sly as the main VAGs primarily studied serotype 2 strains 79 .In fact, the mrp+/epf+/sly+ genotype is almost always reported in serotype 2 strains isolated from diseased pigs in Europe and Asia 34,80 .Moreover, it has been suggested that mrp, epf, and sly are coincidentally associated with virulence rather than being the actual determining factors of virulence, which means the absence of one or more of these proteins does not necessarily result in lack of virulence 30,81 .A more recent whole genome and pan-genome analyses suggested that ofs (encoding for serum opacity factor) and srtF (encoding for sortase F) are stronger predictors for differentiating pathogenicity compared to 71 other previously suggested VAGs in S. suis, including mrp, epf, and sly 82 .
Among the 145 S. suis isolates that were examined by MLST, no previously known STs were found.These isolates were submitted to pubMLST and were assigned as 70 novel STs.Generally, S. suis isolates from clinically healthy pigs are not subjected to MLST, which could partially account for the numerous novel STs in this study 83 .Of note, two specific STs were found to have connections to STs from other countries.ST2157 isolates, which were recovered from the dead piglet, was linked to ST14 from Spain, a serotype 3 strain isolated from a lung tissue showing pneumonia.The diseased piglet was not reported to exhibit manifestations of pneumonia but was only noted to have symptoms of fever before sudden death.On the other hand, ST2161 was connected to ST848 from the United States of America (USA), which was isolated from a pig in 2015.Considering these observed connections, it is tempting to hypothesize if importation and international trade could also be important factors to account for the diversity of S. suis in the Philippines, given that pork and pig products are also common sources of S. suis infections 84 .Based on the data obtained from the Foreign Agricultural Service (FAS) under the US Department of Agriculture (USDA), the Philippines has maintained a longstanding practice of importing live pigs from the United States, with a cumulative total of pigs imported at 14,689 heads from 1967 to 2022.The Philippine Statistics Authority (PSA) reported that a total of 2,703 live pigs were imported mainly from Canada, Spain, Netherlands, Brazil, and the USA 2018 to 2020.The problem, however, is that there is no information on the movement of these imported animals within the Philippines making it impossible to prove the connection of importation and the observed relationship among Philippine isolates sharing a clonal complex with isolates from other countries.
Pending the availability of information on the predominant serotypes of S. suis from clinical cases of infections, the current results, considering the observed predominant serotypes and the entirely novel set of MLST, suggest a potentially different epidemiology of S. suis in the Philippines 34,41,49,38 .
Smallhold farms contribute about 80% of the total hog production in the Philippines.Despite this, the scant information on important bacterial pathogens, especially those that pose zoonotic risks like S. suis, coupled with the limited implementation and understanding of biosecurity-related practices and measures, point to an increased danger of transmission of the pathogen among animals, and at the same time, from animals to humans.
Altogether, the current findings, which provided detailed molecular characterization of circulating S. suis strains in the Philippines for the first time, are highly relevant to both veterinary and human medicine, especially for individuals with direct contact with pigs and pork products, including pig farmers, abattoir workers, as well as the general consumers 3,12,28,35,61 .However, the current study is not without limitations, which must be given attention with further research studies.Particularly, subsequent studies may focus on clinical isolates of S. suis, including historical isolates (if present in regional or national diagnostic laboratories) and newer isolates recovered from more recent cases.Thus, this also emphasizes the importance of continuous surveillance of this pathogen among farms, and even among slaughterhouses and markets in the country, as performed in other countries before 35,36,60 .Also, in consideration to the growing number of studies indicating the propensity of S. suis for rapid development of multidrug resistance 27,[85][86][87][88][89][90][91][92][93][94][95] , characterization of local isolates must also be extended to cover phenotypic and genotypic antimicrobial resistance characteristics to gain an understanding on the applicability and efficacy of currently implemented practices regarding antibiotic usage.Finally, whole genome sequencing and related bioinformatics studies on local isolates, particularly those that were identified as epidemiologically or clinically significant, would further bolster grounded local understanding of this important pathogen.
With plans to further intensify the swine raising industry in the Philippines, the current study provides a snapshot of the on-going conditions related to S. suis infection in key hog-raising provinces in the country.This study functionally marks the national baseline knowledge on S. suis in Philippines.

Ethics statement
Ethics approval for the research, specifically for the conduct of and the protocol for sample collection from farm animals, was obtained from the Institutional Animal Care and Use Committee (IACUC) of the University of the Philippines Los Baños, with the assigned protocol number BIOTECH-2021-001.Additionally, the collection of samples for this study was permitted by each farm owner prior to the sample collection from their pigs.All experiments conducted in this study are in accordance with the relevant regulations, guidelines, and protocols, as evaluated and approved by the UPLB-IACUC.www.nature.com/scientificreports/

Sampling collection
The Philippines is generally divided into three major island groups (Luzon, Visayas, and Mindanao), further subdivided into regions and provinces.While the specific relationships between and among provinces within and among these island groups in relation to trade and transport of live and slaughtered pigs are unknown, it is assumed that a significant percentage of these movements are regional 96 .Using a multistage stratified random sampling approach, it was determined that a minimum of 648 farms were to be sampled based on Cochran's method (1977) with the following assumptions: assumed prevalence (P): 0.3, confidence level (1-α): 0.95, margin of error (α): 0.5, and design effect (DE): 2.0 97 .
Based on the Philippine Statistics Authority's Swine Situation Report as of January 1, 2019 98 , provincial pig population densities were determined, and high hog production provinces were identified.A random sample of three high hog production provinces was selected from each of the three geographical island groups.The provinces randomly selected were Marinduque, Albay, and Batangas for Luzon, Iloilo, Bohol, and Cebu for Visayas, and Misamis Occidental, Zamboanga del Norte, and Misamis Oriental for Mindanao.
Provincial government authorities were requested for a list of villages or barangays that have at least 24 small hold farms.Within these provinces, a random sample of three villages or barangays was selected, and a random sample of 24 households/backyard farms was selected from each barangay.Up to three samples per age group of pigs were collected whenever possible.A sampled pig was classified as either a sow, boar, piglet, or grower by adapting local classification and farming practice.Young pigs housed with the sow and feeding on her milk are considered piglets.A pig housed separately from the sow (i.e., weaned) is considered a grower since distinction among weaners, growers, and finishers is rarely locally applied.
The sampled pigs were manually restrained, and a nose snare and a mouth gag were used to open the mouth and prevent biting.Oral swab samples were collected by rubbing a cotton swab against the oral cavities of the pigs, particularly on the surface of the palatine tonsils whenever safely possible, following the approved protocol identified above.The collection of the swab samples was performed under the guidance of provincial/municipal veterinarians or veterinary technicians.Samples were immediately placed in transport tubes containing 4.0 mL of Amies transport medium.All samples were stored and transported in insulated coolers and immediately processed for streptococcal isolation afterward.

Isolation and Identification
Serial dilutions of the transport medium were prepared, and 100 µL aliquots of varying dilutions were spread plated onto Columbia Agar plates with 5% defibrinated sheep blood.The plates were then incubated at 37 °C under 5% CO 2 for 18-24 h.Up to eight α-hemolytic colonies were selected and purified from each sample [25][26][27][28][29]36,37 . All sccessfully purified isolates were identified using a conventional PCR assay utilizing S. suis-specific recN gene primers, as described elsewhere 99 .
Partial 16 s rRNA gene sequences of selected isolates were determined by Sanger sequencing following the amplification of the said gene using the F1/R13 primers, as described elsewhere 100 .Sequences were submitted to the NCBI Genbank database with accession numbers OR287118-OR287128.To better determine sequence similarity, the 16 s rRNA gene sequences of reference isolates of relevant Streptococcus species 101 were also retrieved from the GenBank.Sequences obtained from this study and the retrieved representative sequences from NCBI were aligned using ClustalW method in Molecular Evolutionary Genetics Analysis (MEGA-X) software 102 .Neighbor-joining method 103 with bootstrap analysis of 1000 replications was used to construct a phylogenetic tree.

Molecular characterization
Further characterization of the identified S. suis isolates was conducted using serotyping, virulence gene profiling, and multilocus sequence typing (MLST).The serotyping of all identified S. suis isolates was carried out using four multiplex PCR assays 19 , all targeting capsular polysaccharide synthesis (cps) genes located on a single locus in the S. suis chromosome.These assays can identify the currently recognized 29 S. suis serotypes.
All S. suis isolates were similarly profiled for the presence of virulence-associated genes using a multiplex PCR assay that targets the extracellular protein factor (epf), muramidase-release protein (mrp), and suilysin (sly) 104 , commonly referred to as virulence-associated genes (VAGs).The MLST was done using multiplex PCR protocol targeting seven housekeeping genes, including aroA (EPSP synthase), cpn60 (60-kDa chaperonin), dpr (peroxide resistance), gki (glucose kinase), mutS (DNA mismatch repair enzyme), recA (homologous recombination), and thrA (aspartokinase) 20 .The PCR products were subsequently submitted for sequencing.One hundred twenty-five serotypeable isolates were sequenced for MLST.The remaining six serotypeable isolates did not yield amplicons for one or more of the target genes and thus, were not typed by MLST.An additional 20 nontypeable isolates were selected in random and similarly sequenced.Recovered sequences were submitted to pubMLST (March-May 2023) for verification and typing assignment.The complete dataset pertaining to the isolates, typed by MLST, including provenance/relevant metadata and sequences, can be accessed through the pubMLST database under ID numbers 3539-3546, 3550-3606 and 3660-3739.
The entire S. suis database in pubMLST (available on August 13, 2023) was downloaded and used to visualize and identify groups of related genotypes and clonal complexes using the goeBURST algorithm in the PHYLOViZ software 21 .

Statistical analysis
Data were stored in an in-house Excel 365 (Microsoft) database.Descriptive statistics related to isolation rates per province, farm, and age group as well as the proportion of each serotype among the isolates, were calculated using the same software.Confidence interval estimates of the proportion were calculated with a confidence level

Figure 1 .
Figure 1.Geographical distribution of the recovered isolates of Streptococcus suis among nine provinces in the Philippines.The map was created using MapChart software (https:// www.mapch art.net).

Figure 2 .
Figure 2. Phylogenetic relationship of recovered Streptococcus suis isolates and representative reference streptococcal strains, as inferred from the 16 s rRNA gene sequence comparisons using the neighbor-joining method.